Project Summary Autoimmune Polyglandular Syndrome type 1 (APS-1) is caused by mutations in the Autoimmune Regulator (Aire) gene, which normally promotes central and peripheral T cell tolerance. APS-1 patients exhibit autoimmune disorders in multiple organs and a predisposition to chronic mucocutaneous candidiasis (CMC). A lack of mechanistic under- standing of the basis of candidiasis and its paradoxical association with autoimmunity hampers effective treatment of APS-1. Patients with candidiasis often have defects in cell-mediated immunity. APS-1 patients produce class- switched IgG autoantibodies against T helper 17 (TH17) cytokines, which may neutralize and impair protective anti- fungal immunity. This highlights humoral immune dysfunction as a potentially important contributing factor to CMC in APS-1. Our long-term goal is to understand the immune dysregulation in autoimmune and immunodeficiency disor- ders and to develop mechanism-guided therapeutic strategies. The objective in this application is to elucidate the mechanism of humoral immune defects in APS-1. Our preliminary studies show that AIRE is expressed specifically in human and mouse germinal center (GC) B cells in secondary lymphoid organs in vivo, and in B cells undergoing antibody diversification in vitro. AIRE deficiency in B cells causes elevated class switch recombination (CSR) and somatic hypermutation (SHM). AIRE suppresses CSR when introduced into AIRE-deficient B cells. AIRE interacts in B cells with activation-induced cytidine deaminase (AID), the enzyme essential for SHM and CSR. Aire?/? mice have aberrant expansion of GC B cell and T cell responses in systemic and mucosal lymphoid tissues. These findings support the central hypothesis that APS-1 involves aberrantly increased antibody maturation caused by B cell- intrinsic defects in AIRE-mediated AID regulation and B cell-extrinsic defects in the selection against autoreactive antibodies due to excessive GC response. Our rationale is that the elucidation of the mechanism of humoral immune defects in APS-1 will prompt more specific treatment of APS-1. The hypothesis will be tested by pursing two specific aims. Employing quantitative real-time PCR, flow cytometry, chromatin immunoprecipitation, genomic uracil dot blot and next-generation sequencing, studies in Aim 1 will determine the abnormalities in antibody maturation and the antibody repertoire in APS-1 and the mechanism by which AIRE regulates AID in B cells. Studies in Aim 2 will use competitive ELISA as well as mouse models of cutaneous Candida albicans infection to determine the role of B cell- intrinsic and -extrinsic AIRE deficiency in APS-1-associated candidiasis. This study proposes innovative and cutting- edge methodologies to test novel and mechanistic hypotheses in APS-1 pathogenesis. The work will have sustained and broad impact on the field of basic and clinical immunology, as it will not only advance the understanding of the disease APS-1 and AID-mediated regulation of antibody maturation, but also offer insights into the enigmas of an increasing number of immunodeficiencies that co-present with autoimmunity, thus paving the way to better treat the- se diseases.